The cloning of DNA segments is performed as a daily routine in many research labs; it is frequently also a prerequisite step in genetic analyses or the preparation of DNA constructs for gene expression or regulation. A great deal of time and effort can be expended in the cloning of DNA segments. The basic methods for cloning have been known for many years and have changed little during that time. A typical cloning protocol based on restriction enzyme digestion is as follows: (1) digest the DNA to be cloned with restriction enzymes; (2) purify the digested DNA segment to be cloned; (3) prepare the vector by cutting with appropriate restriction enzymes, treating with alkaline phosphatase, purifying etc., as appropriate; (4) ligate the DNA segment to the vector, with appropriate controls to estimate background of uncut and self-ligated vector; and (5) introduce the resulting vector into a host cell.
Restriction enzyme based cloning has the disadvantage that the DNA to be cloned must not contain sequences within it that are the same as the sequences recognized by the restriction enzymes with which it will be digested. This is often inconvenient, in some cases it may not even be known whether these sites are present within the DNA segment to be cloned since it is often desirable to clone a DNA segment without their full sequences being known; for example DNA segments may be amplified by the polymerase chain reaction when only the sequences at their ends are known. Restriction enzyme based cloning is also time consuming, with the restriction digestion, purification and ligation steps requiring significant incubation times (˜1 hour each).
Some of these limitations have been addressed with the introduction of vectors that contain a 3′ overhang of a single T residue (Cha et al., 1993. Journal/Gene, 136: 369-370; Ichihara and Kurosawa, 1993. Journal/Gene, 130: 153-154; Ido and Hayami, 1997. Journal/Biosci Biotechnol Biochem, 61: 1766-1767; Zhou and Gomez-Sanchez, 2000. Journal/Curr Issues Mol Biol, 2: 1-7.). These vectors allow inserts with a 3′ overhang of a single A residue to be ligated in without the need for restriction digestion. However this procedure requires that the DNA segment to be cloned is amplified with a low fidelity non-proof-reading polymerase, or that a second step subsequent to amplification is performed to add the 3′ A residue. The slow ligation step is also still required.
Cloning methods have also been developed in which recombinases are used in vitro to combine vector and insert DNA (Hartley et al., 2000. Journal/Genome Res, 10: 1788-1795.). In this case the DNA segment to be cloned requires the addition of ˜25 bp sequences to each end to allow recognition by the recombinase. Again a slow step (˜1 hour) is also required, this time to allow the recombinase to act.
An extremely powerful molecular cloning method using vaccinia DNA topoisomerase has been described (Cheng et al., 1998. Journal/Cell, 92: 841-850; Geng et al., 2006. Journal/Mol Biotechnol, 33: 23-28; Heyman et al., 1999. Journal/Genome Res, 9: 383-392; Shuman, 1994. Journal/J Biol Chem, 269: 32678-32684; Shuman, 1998. Journal/Mol Cell, 1: 741-748.) and U.S. Pat. Nos. 5,766,891; 6,548,277; 6,653,106; 6,916,632 and 7,026,141. This method avoids the limitations of alternative cloning methods: PCR amplification products need no modification and the cloning reaction is extremely short (˜5 minutes).
Previously described cloning methods using vaccinia topoisomerase require the reaction of a donor DNA molecule with vaccinia DNA topoisomerase enzyme. A tyrosyl residue in the protein (Tyr-274) forms a covalent adduct with the 3′ phosphate of the fifth base of a consensus pentapyrimidine element (C/T)CCTT (Shuman, 1991. Journal/J Biol Chem, 266: 1796-1803; Shuman and Prescott, 1990. Journal/J Biol Chem, 265: 17826-17836.) in the donor DNA molecule. This reaction is reversible. When the scissile bond is situated close to (within approximately 10 bp of) the 3′ end of the DNA duplex, the downstream portion of the cleaved strand spontaneously dissociates, preventing reversal of the reaction and forming a stable covalent adduct. This covalent adduct is then purified. When the covalent adduct is mixed with an acceptor DNA molecule, the topoisomerase that is covalently bound to the donor molecule catalyzes the joining of the two DNA molecules. This joining requires that the 5′ ends of the acceptor DNA are hydroxylated (not phosphorylated) and that the single-stranded overhangs of the donor and acceptor, if any, are compatible (i.e. complementary).
Topoisomerase-based cloning is in principle a powerful cloning technology, allowing the rapid cloning of any blunt or sticky-ended DNA fragment (Geng et al., 2006. Journal/Mol Biotechnol, 33: 23-28.). However the previously described methods require extensive preparation and processing steps to make a vector suitable for topoisomerase-based cloning. Vaccinia topoisomerase only cleaves one of the DNA strands of the donor molecule. For the donor to be ligated to the acceptor DNA molecule by vaccinia topoisomerase, however, both strands of the donor must be cleaved. This can be accomplished by ligating oligonucleotides containing the consensus pentapyrimidine element (C/T)CCTT to a DNA molecule that has been cleaved with a restriction endonuclease. The ligation mixture is then purified, re-cut and re-purified. Treatment of the DNA with purified topoisomerase (and additional oligonucleotides) produces a covalent adduct between the DNA and the topoisomerase. This adduct is then purified to produce the donor DNA (Heyman et al., 1999. Journal/Genome Res, 9: 383-392.) (see U.S. Pat. Nos. 5,766,891; 6,548,277; 6,653,106; 6,916,632 and 7,026,141).
The multiple processing and purification steps required to convert a vector into a topoisomerase-adduct donor molecule limit the ease with which topoisomerase-based cloning can be applied to new vectors. There is therefore a need in the art for a cloning method that allows simple cloning of any DNA molecule without the extensive preparation required by current methodologies. These and other unmet needs are provided by the presently claimed methods, compositions, and kits.